Purpose:
Although bio-engineered limbal epithelial cell sheets have showed a good results for severe limbal deficiency patients, some of cases are difficult to improve visual acuity caused by anterior stromal haziness. To treat for the patients, we have developed bio-engineered epithelio-stromal lenticule. The aim of this study is to determine the corneal lenticule using rabbit experimental model.

Methods:
Human corneal lenticules were decellularized by 0.25% trypsin-EDTA in hypotonic Tris buffer (10mM, pH 7.2) and human corneal limbal epithelial cells were re-seeded on the lenticule. Limbal deficient experimental model was established by alkali burn with 1N NaOH and decellularized and bio-engineered lenticule was transplantated in the burned site after 1 week. The operated corneas were collected at 1 week and 4 weeks. Limbal deficiency and invasion of conjunctival cells were examined by impression cytology and routine histology, and harvested corneas were analyzed by immuonohistochemistry.

Results:
Immunologic staining results showed that human cornea lenticules were successfully decellularized and limbal epithelial cells reseeded expressing Keratin 3, 5, 12, and ABCG2. Impression cytology after alkali burn showed numerous goblet cells at inside of limbus, and several neovascularization were observed. Transplantation of the lenticules showed that the lenticule with limbal epithelial cells inhibited conjunctival cell migration into center cornea compare to lenticule only, and had more transparent than the other at 4 weeks after operation.

Conclusions:
Data in this study showed that bio-engineered lenticules is more suitable for improving limbal deficiency than decellularized lenticules only, and corneal epithelial cells were remained and kept its phenotype without any infiltration of inflammatory cells. Collectively, our data suggest that bio-engineered human cornea lenticule has great potential for the replacement of allogenic cornea transplantation to treat severe corneal diseases.